It is the long term goal of this research to identify and characterize at a molecular level, transport proteins and receptors in corneal cells which contribute to maintenance of corneal deturgescence and transparency. This includes determining their primary amino acid sequence, prevalence, regulation, specific mechanisms of function and relative role in the maintenance of the health of corneal cells. Four specific aims are proposed: l) Using microdissection, molecular biology, and patch voltage clamping, determine the location of ion channels in both corneal epithelium and endothelium. 2) For corneal epithelium, determine the relative role of its major ion channels in controlling the transmembrane voltage using molecular biology, ratio imaging, cell culture, and patch voltage clamping. This includes determining the primary amino acid sequence of the potassium channels and non-selective cation channels and how each channel is regulated. 3) For corneal endothelium, determine the relative role of its major ion channels in controlling the transmembrane voltage using molecular biology, ratio imaging, cell culture, and patch voltage clamping. This includes determining the primary amino acid sequence of the potassium channels and non-selective cation channels and how each channel is regulated. 4) Compare channel sequences in corneal cells to those in ciliary epithelium and conjunctival epithelium. 5) Characterize the channel and receptor proteins in corneal keratocytes using molecular biology, cell culture patch voltage clamping, and imaging. Determine whether or not the cells contain muscarinic receptors and if so what types. Sequence the channel proteins and determine their sensitivity to toxins and Prozac. Quantify dye and electrical coupling between the cells. Polymerase chain reaction and DNA sequencing techniques will be used to determine the primary amino acid sequence of each transporter or receptor. Site directed mutagenesis and transfections into expression systems will be used along with patch voltage clamp to measure at a whole cell and single channel level the current flow through these transporters to assess structure-function relationships for each as well as to determine how each is regulated. The roles of selected drugs and/or antibodies in regulating transporter function will be investigated.